Monocyte-origin multipotent cell MOMC

ABSTRACT

The present invention is to provide a multipotent cell wherein the sufficient amount necessary can be stably and conveniently supplied with a minimum invasion, that will not cause rejection at the time of cell transplantation, that has a potential to differentiate into various cells such as mesenchymal cells including bone, cartilage, skeletal muscle and fat, endothelial cells, myocardial cells, neurons, mesenchymal cells, myocardial cells, endothelial cells, neurons induced to differentiate from the multipotent cell, and a therapeutic agent/treating method comprising these as active ingredient. Peripheral blood mononuclear cells (PMBC) are cultured on fibronectin-coated plastic plates for 7 to 10 days. The generating cell population with a fibroblast-like morphology is derived from circulating CD14 +  monocyte, with a unique phenotype of CD14 + CD45 + CD34 +  type I collagen + . These cells have a potential to differentiate into mesenchymal cells including bone, cartilage, skeletal muscle and fat, endothelial cells, myocardial cells, and neurons under particular culture conditions.

TECHNICAL FIELD

The present invention relates to a monocyte-derived multipotent cell ofCD14⁺ and CD34⁺ that can differentiate into mesoderm cells such asmesenchymal cells, myocardial cells, and endothelial cells or intoneurons; a mesodermal cell/tissue such as mesenchymal cell, myocardialcell and endothelial cell, and neuron/nerve tissue induced todifferentiate from the monocyte-derived multipotent cell; a therapeuticagent comprising these as active ingredient; and to a treating methodcomprising administering these.

BACKGROUND ART

Peripheral blood monocytes are derived from bone marrow hematopoieticstem cells and are known to differentiate into several phagocytes,including macrophages, dendritic cells, osteoclasts, microglial cells ofcentral nervous system, and liver Kupffer cells (Bioessays, 17, 977-986,1995; Blood, 98, 2544-5254, 2001; BMC Immunol., 3, 15, 2002; Microsc ResTech., 39, 350-364, 1997). The differentiation of the monocytes intovarious phagocytes is controlled by signaling of various growth factors.In other words, differentiation into macrophages, dendritic cells,osteoclasts are controlled by signaling of M-CSF, GM-CSF and IL-4, andreceptor activating factors of NF ligand or M-CSF, respectively (see forexample, Blood, 98, 2544-5254, 2001; BMC Immunol., 3, 15, 2002; J ExpMed., 190, 1741-1754, 1999). Until recently, it was believed that thedifferentiation potential of monocytes was restricted to phagocytes.However, recent studies have shown that human monocytes candifferentiate into endothelial-like cells by culturing in vitro with acombination of angiogenic factors (see for example, Differentiation, 65,287-300, 2000; Cardiovascular Res., 49, 671-680, 2001). In addition, theexpression of bone-specific alkaline phosphatase was reported during themonocyte differentiation process in the in vitro granuloma model (seefor example, Immunobiology, 202, 68-81, 2000). However, thedifferentiation potential is not completely clarified and it was notknown whether monocytes had differentiation potential into cell typesother than phagocytes.

On the other hand, it was revealed that many adult tissues containpopulations of stem cells that can self-replicate and give rise todaughter cells that undergo an irreversible terminal differentiation(see for example, Science, 287, 1442-1446, 2000). The best-characterizedare hematopoietic stem cells and their progeny, but stem cells areidentified in most of the tissues, including mysenchymal, neuron, andhemotopoietic cells (see for example, Science, 284, 143-147, 1999;Science, 287, 1433-1438, 2000; J. Hepatol., 29, 676-682, 1998).Mesenchymal stem cells (MSCs) are identified as adherent fibroblast-likecells in the bone marrow with differentiation potential into mesenchymaltissues, including bone, cartilage, fat, muscle, and bone marrow stroma(see for example, Science, 284, 143-147, 1999). Recently, mesenchymalprogenitors having morphologic and phenotypic features anddifferentiation potentials similar to MSCs have been reported atextremely low frequencies in umbilical cord blood (see for example, Br.J. Haematol., 109, 235-242, 2000), as well as in fetal (see for example,Blood, 98, 2396-2402, 2001) and adult peripheral blood (see for example,Arthritis Res., 2, 477-488, 2000). However, MSCs and circulatingMSC-like cells do not express various hematopoietic markers or the stemcell/endothelial marker CD34 (see for example, Science, 284, 143-147,1999; Br. J. Haematol., 109, 235-242, 2000; Blood, 98, 2396-2402, 2001).

As described above, various postnatal tissue-specific stem cells andembryonic stem (ES) cells are currently being analyzed as candidatesources for future therapeutic intervention for tissue regeneration (seefor example, Science, 287, 1442-1446, 2000). It has been reported thatbone marrow-derived MSCs engraft in many organs and differentiate alongtissue-specific lineages upon its transplantation in animal models (seefor example, Nat. Med., 6, 1282-1286, 2000; Science, 279, 1528-1530,1998), as well as in human infant suffering osteogenesis imperfecta (seefor example, Nat. Med., 5, 309-313, 1999). However, MSCs are rare inadult human bone marrow (0.01% to 0.001%), and expansion of MSCs to thenumber of cells required for regeneration therapy is technicallydifficult, expensive, and time-consuming (see for example, Stem Cells,19, 180-192, 2001). ES cells are multipotent cells derived from germinalcells that can be propagated indefinitely in vitro being stillundifferentiated and induced to differentiate to most cell types in vivo(see for example, Trends Biotechnol., 18, 53-57, 2000). Although EScells have been isolated from human, their use in research as well astherapeutic is cumbered by ethical considerations (see for example,Science, 287, 1397, 2000).

Several different precursors that can differentiate into endothelial ormesenchymal cell types have been reported in human postnatal peripheralblood, including endothelial cells (see for example, Science, 275,964-967, 1997), smooth muscle cells (see for example, Circulation, 106,1199-1204, 2002), and mesenchymal cells (see for example, ArthritisRes., 2, 477-488, 2000). In vitro expansion of endothelial and smoothmuscle progenitors requires a combination of several growth factors (seefor example, Science, 275, 964-967, 1997; Circulation, 106, 1199-1204,2002). Mesenchymal progenitors can be expanded in a medium supplementedwith 20% fetal bovine serum (FBS) without any additional growth factors,but their development in PBMC cultures was reported to be unaffected byeliminating CD14⁺ cells (see for example, Arthritis Res., 2, 477-488,2000). However, these endothelial or mesenchymal cells do not have thephenotypic characteristics to be positive to CD 14, CD45, CD34 and typeI collagen.

The big object remaining in modern medicine is said to overcomedeficiency of organs due to disease or external injuries or functionalimpairment. The only method that can be practiced today for treatingsuch condition is organ transplantation. However, there are still manydifficulties for spreading as an actual treating method, due to problemssuch as brain-death diagnosis or supply from donors. On the other hand,regenerative medicine intending regeneration of organs draws attentionwith the recent development of stem cells and developmental biology, andis expected as the direction of the medicine to advance in the 21stcentury. In animal models, functional recoveries of organs bytransplantation of ES cells have been reported, while the application inhuman is stuffed due to rejection or ethical problems of the use of EScells. Further, as various adult tissues stem cells (mesenchymal, bloodvessels, liver etc.) are extremely few in vivo, the isolation thereof istechnically difficult, and it is hard at the present time to obtainsufficient amount of cells for transplantation. Therefore, there aremany problems to be solved before the regenerative medicine using EScells or tissue stem cells can be applied to the actual medicine.Particularly, it is essential to supply cells having differentiationpotential in a stable manner so that regenerative medicine becomes areality.

The object of the present invention is to provide a multipotent cellthat can differentiate into various cells such as mesenchymal cellsincluding bone, cartilage, skeletal muscle and fat, endothelial cells,myocardial cells and neurons wherein a sufficient amount can be suppliedstably with minimum invasion, without problems such as securing donorsand rejection in cell transplant, and with less ethical considerations;a mesodermal progenitor/mesodermal cell/mesodermal tissue and aneuron/nerve tissue, such as mesenchymal cells, myocardiac cells,endothelial cells, being induced to differentiate from themultipotential cell; a therapeutic agent comprising these as activeingredient; and a treating method administering the same.

The present inventors confirmed the expression of fibroblast-like cellswhen peripheral blood mononuclear cells (PBMCs) are cultured onfibronectin-coated plastic plates for 7 to 10 days. Being interested bythe origin and physiological function of this human cell populationexhibiting a fibroblast-like morphology, the present inventors foundthat these cells are derived from circulating CD14⁺ monocytes, with aunique phenotype of CD14⁺CD45⁺CD34⁺ type I collagen⁺, and having apotential to differentiate into mesenchymal cells including bone,cartilage, smooth muscle and fat, endothelial cells, myocardial cells,neurons, under a particular culture condition. They named this cell themonocyte-derived multipotent cell (MOMC). With the knowledge havingrevealed for the first time that circulating monocytes are multipotentprogenitors having a differentiation potential not only into phagocytesbut also into various mesenchymal cells, the present inventors have thuscompleted the invention.

DISCLOSURE OF THE INVENTION

In other words, the present relation relates to: a monocyte-derivedmultipotent cell, derived from a monocyte, which expresses CD14 and CD34 (“1”); a monocyte-derived multipotent cell, derived from a monocyte,which expresses CD14, CD34, CD45 and type I collagen (“2”); themonocyte-derived multipotent cell according to “1” or “2”, that candifferentiate into mesenchymal cells by a culture under a conditioninducing differentiation into mesenchymal tissues (“3”); themonocyte-derived multipotent cell according to “3”, wherein themesenchymal cells are osteoblasts, skeletal myoblasts, chondrocytes oradipocytes (“4”); the monocyte-derived multipotent cell according to “1”or “2”, that can differentiate into myocardial cells by a culture undera condition inducing differentiation into cardiac muscle such as acoculture with cultured myocardial cells (“5”); the monocyte-derivedmultipotent cell according to “1” or “2”, that can differentiate intoneuron by a culture under a condition inducing differentiation intonerve, such as a coculture with cultured neuron (“6”); themonocyte-derived multipotent cell according to “1” or “2”, that candifferentiate into endothelial cells, by a culture under a conditioninducing differentiation into endothelium such as a culture under acondition maintaining endothelial cells (“7”); and the monocyte-derivedmultipotent cell according to “1” or “2”, that can differentiate intomesodermal cells (“8”).

Furthermore, the present invention relates to a method for preparing amonocyte-derived multipotent cell, comprising culturing peripheral bloodmononuclear cells (PBMCs) in vitro on fibronectin, and collectingfibroblast-like cells expressing CD14 and CD34 (“9”); the method forpreparing a monocyte-derived multipotent cell according to “9”,comprising culturing in vitro on fibronectin for 5 to 14 days (“10”); amesenchymal progenitor, a mesenchymal cell or a mesenchymal tissueinduced by culturing the monocyte-derived multipotent cell according toany one of “1” to “8”, under a condition inducing differentiation intomesenchymal tissues (“11”); the mesenchymal progenitor, the mesenchymalcell or the mesenchymal tissue according to “11”, wherein themesenchymal cells are osteoblasts, skeletal myoblasts, chondrocytes oradipocytes (“12”); a myocardial progenitor, a myocardial cell or amyocardial tissue induced by culturing the monocyte-derived multipotentcell according to any one of “1” to “8”, under a condition inducingdifferentiation into cardiac muscle such as a coculture with culturedmyocardial cells (“13”); a neural progenitor, a neuron or a nerve tissueinduced by culturing the monocyte-derived multipotent cell according toany one of “1” to “8”, under a condition to inducing differentiationinto nerve, such as a coculture with cultured neuron (“14”); anendothelial progenitor, an endothelial cell or an endothelial tissueinduced by culturing the monocyte-derived multipotent cell according toany one of “1” to “8”, under a condition inducing differentiation intoendothelium, such as a culture under a condition maintaining endothelialcells (“15”); a mesodermal progenitor, a mesodermal cell or a mesodermaltissue induced to differentiate from the monocyte-derived multipotentcell according to any one of “1” to “8” (“16”).

Moreover, the present invention relates to a therapeutic agentcomprising as active ingredient the monocyte-derived multipotent cellaccording to any one of “1” to “8” and/or mesodermal progenitors,mesodermal cells and/or mesodermal tissues induced to differentiate fromthe monocyte-derived multipotent cell (“17”); or a therapeutic agentcomprising as active ingredient the monocyte-derived multipotent cellaccording to any one of “1” to “8” and/or neural progenitors, neuronsand/or nerve tissues induced to differentiate from the monocyte-derivedmultipotent cell (“18”); a treating method comprising administering themonocyte-derived multipotent cell according to any one of “1” to “8”and/or mesodermal progenitors, mesodermal cells and/or mesodermaltissues induced to differentiate from the monocyte-derived multipotentcell (“19”); or a treating method comprising administering themonocyte-derived multipotent cell according to any one of “1” to “8”and/or neural progenitors, neurons and/or nerve tissues induced todifferentiate from the monocyte-derived multipotent cell (“20”).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-G show the morphology of MOMCs of the present invention. (FIG.1A, FIG. 1B) PBMCs were cultured on fibronectin in low-glucose DMEMsupplemented with 10% FBS for 7 days, and observed with a phase contrastmicroscope. The original magnifications are ×80 for FIG. 1A, and ×40 forFIG. 1B. MOMCs were moved on a new fibronectin-coated plate and culturedfor 24 hours. FIG. 1C shows the observation results with a phasecontrast microscope (×40). FIGS. 1D-E show observation results with anelectron microscope (FIG. 1D and FIG. 1E, ×5000; FIG. 1F and FIG. 1G,×30000). A bundle of intermediate filaments is shown by an arrow, and astructure similar to a rod-shaped microtubulated body is shown by anarrowhead. L is for labyrinth-like endocytic vesicle; LD is for lipiddroplet; N is for nucleus, and PS is for pseudopodia.

FIG. 2 shows the result of flow cytometric analysis of MOMCs of thepresent invention. PMBCs were cultured on fibronectin-coated plates andthe adherent cells were harvested on Day 7. The cells were stained witha series of mAbs shown in the picture and analyzed by flow cytometry.The expression of the molecule of interest is shown as shadedhistograms. Open histograms represent controls stained withisotype-matched control mAbs. The results shown are representative of atleast three experiments.

FIGS. 3A-D demonstrate that MOMCs of the present invention originatefrom circulating CD14+ monocytes. (FIG. 3A) PBMCs were cultured onfibronectin for 1 hour, and 1, 3, 5, 7, 10, and 14 days. The adherentcells were harvested and stained with FITC-conjugated anti-CD14 andPC5-conjugated anti-CD34 mAbs and analyzed by flow cytometry. Theresults shown are representative of three independent experiments. (FIG.3B) PBMCs depleted of CD14+ cells, CD34+ cells or CD 105+ cells, andmock-treated PMBCs were cultured on fibronectin for 7 days. The numberof attaching cells per cm3 was counted, and the results are expressed asthe ratio to the number of attached cells in the untreated PBMC culture.The results shown are the mean and SD from three donors. The asterisk inthe figure indicates a significant difference compared with mock-treatedPBMC cultures. (FIG. 3C) MACS (Magnetic Cell Sorting)-sorted CD14+monocytes were stained with PKH67 and cultured with or without unlabeledCD14− cells (ratio 1:4) on fibronectin-coated or uncoated plastic platesfor 7 days. The adherent cells were harvested, stained withPC5-conjugated anti-CD34 mAb, and analyzed by flow cytometry. Theresults shown are representative of three experiments. (FIG. 3D)MACS-sorted CD14+ monocytes were stained with CFSE and cultured onfibronectin for 0, 1, 3, and 5 days. The adherent cells were harvestedand stained with PC5-conjucated anti-CD14 mAb. The cells were analyzedby flow cytometry. The results shown are representative of threeexperiments.

FIGS. 4A-H show the result of immunohistochemical analysis of MOMCs ofthe present invention. MOMCs generated by culturing PBMCs onfibronectin-coated plates for 7 days were moved onto fibronectin-coatedchamber slides (The slides coated with type I collagen were only usedfor fibronectin staining). After 24 hours of culture, theabove-mentioned slides were fixed with 10% formalin and stained withmAbs as indicated in the picture. The nuclei were counterstained withhematoxylin. Original magnification is ×100. The results shown arerepresentative of at least three independent experiments.

FIGS. 5A-C show how the MOMCs of the present invention proliferate.MOMCs generated by culturing PBMCs on fibronectin-coated plates for 7days were moved on fibronectin-coated chamber slides. MOMCs were furthercultured for 1, 3, 5, 7, and 10 days, and stained with BrdU. The nucleiwere counterstained with hematoxylin. (FIG. 5A) A representative figureat Day 1 and Day 5. The arrow indicates nuclei positive for BrdUstaining. (FIG. 5B) At least 200 cells were counted to see the BrdUstaining experiment result and the number of BrdU+ cells was calculatedfor individual slides cultured for 1, 3, 5, 7, and 10 days. The resultsshown are the mean and SD of five independent experiments. (FIG. 5C)MOMCs were labeled with CFSE and were cultured on new fibronectin-coatedplates for 0, 1, 3, 5 days. The adherent cells were collected andanalyzed by flow cytometry. The results shown are representative ofthree independent experiments.

FIGS. 6A-K show osteogenic, myogenic, chondrogenic, and adipogenicdifferentiation of MOMCs of the present invention. MOMCs before andafter three weeks of osteogenic induction were stained with Alizarin red(FIG. 6A; magnification ×100) or with alkaline phosphatase (FIG. 6B;×100). The intracellular calcium deposition was measured in MOMCs andfibroblasts before and after osteogenic induction and expressed asmicrogram per microgram protein content (FIG. 6C). Expression of mRNAsfor osterix, bone sialoprotein II (BSP II), osteocalcin, CD34, CD45,CD14 and GAPDH were examined in MOMCs before and after 3 weeks ofosteogenic induction and in osteosarcoma cell line (FIG. 6D). MOMCsbefore and after 3 weeks of myogenic induction were stained, andSkM-actin staining (FIG. 6E; ×200) or SkM-MHC staining (FIG. 6F; ×200)were examined. Expression of mRNAs for myogenin, SkM-MHC, CD34, CD45,CD14, and GAPDH was examined in MOMCs before and after 3 weeks ofmyogenic induction and in myoblast, muscle tissue, and inrhabdomyosarcoma cell line (FIG. 6G). MOMCs before and after 3 weeks ofchondrogenic induction were stained and type II collagen staining wereexamined (FIG. 6H; ×40). Expression of mRNAs for α1 (type II) and α1(type X) collagen, CD34, CD45, CD14, and GAPDH was examined in MOMCsbefore and after 3 weeks of chondrogenic induction and in achondrosarcoma cell line (FIG. 6I). MOMCs before and after 3 weeks ofadipogenic induction were stained with Oil-red-O (FIG. 6J; ×200).Expression of the mRNAs for PPARγ, aP2, CD34, Cd45, CD14 and GAPDH wasexamined in MOMCs before and after three weeks of adipogenic inductionand in fat tissue (FIG. 6K). The results shown are representative of atleast five experiments.

FIGS. 7A-H show the coexpression of CD45 (green) and tissue-specifictranscription factors (red) in MOMCs of the present invention thatunderwent 1 week of mesenchymal differentiation. MOMCs before inductiontreatment (FIGS. 7A-D) and MOMCs treated for osteogenic FIG. 7E,myogenic FIG. 7F, chondrogenic FIG. 7G or adiopogenic FIG. 7H inductionfor 1 week were examined for the immunohistochemical localization ofCD45 in combination with Cbfa1/Runx2 (FIG. 7A and FIG. 7E), MyoD (FIG.7B and FIG. 7F), Sox-9 (FIG. 7C and FIG. 7G), or PPARγ (FIG. 7D and FIG.7H). The cells were observed with confocal laser fluorescence microscopy(original magnification ×200). The results shown are representative ofthree experiments.

FIGS. 8A-D show how MOMCs of the present invention differentiate intomyocardium. Nestins (brown) expressed in MOMCs cocultured with Wistarrat-cultured myocardial cells for 8 days were immunostained (FIG. 8A×200). After labeling cell membrane with fluorescent PKH67 (green),MOMCs were cocultured with Wistar rat-cultured myocardial cells for 7days, and were double fluorescently immunostained with Nkx 2.5 (red), amyocardial cell-specific transcription factor, and CD45 (blue), ahematopoietic marker (FIG. 8B ×200). After labeling cell membrane withfluorescent PKH67 (green), MOMCs were cocultured with Wistarrat-cultured myocardial cells for 8 days, and were double fluorescentlyimmunostained with eHAND (red), a myocardial cell-specific transcriptionfactor, and CD45 (blue), a hematopoietic marker (FIG. 8C ×200). MOMCswere cocultured with Wistar rat-cultured myocardial cells for 3, 6, 9,12 days and expression of the mRNAs for myosin light chain (MLC2v), amyocardial cell-structural protein (FIG. 8D). The results shown arerepresentative of three experiments.

FIGS. 9A-E show how MOMCs of the present invention differentiate intoneurons. Nestins (brown), which are markers expressing innerve/myocardial progenitors, and being expressed in MOMCs coculturedwith Wistar rat-cultured neurons for 8 days were immunostained (FIG. 9A×200). After labeling cell membrane with fluorescent PKH67 (green),MOMCs were cocultured with Wistar rat-cultured neurons for 4 days, andwere double fluorescently immunostained with NeuroD (red), aneuron-specific transcription factor, and CD45 (blue), a hematopoieticmarker (FIG. 9B ×200). MOMCs were cocultured with Wistar rat-culturedneurons for 3 days, and were double fluorescently immunostained withnestin (brown) and Neurogenin 2 (red), a neuron-specific transcriptionfactor (FIG. 9C ×200). PKH67-labeled MOMCs (green) were cocultured withWistar rat-cultured neuron for 3 days, and were double fluorescentlyimmunostained with Hu (red), a mature-nerve marker (FIG. 9D ×200).PKH67-labeled MOMCs were cocultured with Wistar rat-cultured neuron for9 days, and were fluorescently immunostained with NeuN (red), amature-nerve marker (FIG. 9E ×200). The results shown are representativeof three experiments.

FIGS. 10A-C show how MOMCs of the present invention differentiate intoendothelial cells. MOMCs induced to differentiate in a EBM-2 medium, amaintenance medium of endothelia cells, for 7 days, changed to amorphology having multiple projections from a spindle shape (FIG. 10A×200). CD34 and endothelial cell-specific vWF, eNOS, VEGFR2/KDR/Flk-1,expressed in MOMCs induced to differentiate in an EBM-2 medium for 7days, were immunostained (brown) (FIG. 10B ×200). Expression of themRNAs for Flt-1, VEGFR2/KDR/Flk-1, CD31, CD144, vWF, CD34, CD45, CD14,and GAPDH, expressed in endothelial cells after 7-day culture of MOMCsin an EBM-2 medium, was examined (FIG. 10C). The results shown arerepresentative of three experiments.

BEST MODE OF CARRYING OUT THE INVENTION

As for the monocyte-derived multipotent cell (MOMC) of the presentinvention, there is no specific limitation as long as it is derived froma monocyte, and it is a cell or a cell population expressing CD14, CD34,CD45 and type I collagen, derived from a cell, a cell population or amonocyte expressing CD14 and CD34. The origin of the above monocytes isnot particularly limited including mice, rats, dogs, pigs, monkey andhuman, and human being preferable. As for human monocyte, it can be amonocyte from a donor, while an autologous one is preferable. As for thesource of monocytes, monocytes derived from peripheral blood or bonemarrow, or monocytes differentiated from hematopoietic stem cells exvivo can be exemplified. The monocytes herein mentioned are defined tobe positive to CD14 or CD11b.

The above-mentioned CD14 and CD45 are known to be a marker of monocyteand cell derived from monocyte, CD34 a marker of endothelial and stemcells, and type I collagen a marker of mesenchymal cells. As for themonocyte-derived multipotent cell of the present invention, thoseexpressing CD105 and Sca-1 as stem cell markers, type III collagen andfibronectin as mesenchymal cell markers, and VE cadherin and Flt-1 asendothelial markers are preferable. The MOMCs are cells distinct frommonocytes, macrophages or dendritic cells from their protein expressingpattern mentioned above, and it can be said that MOMCs are a new cellpopulation having combined the characteristics of mesenchymal cells,endothelial cells and stem cells.

The MOMCs of the present invention are derived from circulating CD14⁺monocytes, as cited in the following. First, MOMCs are positive for themonocyte lineage markers CD14, CD13, CD11b, CD11c and CD64. Second,serial phenotypic analyses of adherent peripheral blood cells culturedon fibronectin showed increased expression of CD34 on the adherent CD14⁺cells. Third, the development of MOMCs was almost completely inhibitedby depleting the PBMCs of CD14⁺ cells, but not by depleting the PBMCs ofcells positive for CD34 or CD105. Finally, studies using labeled-CD14⁺cells revealed up-regulated expression of CD34 on CD14⁺ monocytes, andthat there was no cell proliferating rapidly in PBMC cultures among theCD14⁻ cell population.

As for the MOMCs of the present invention, a cell with multipotency thatcan differentiate into mesodermal cells under induction condition knownto differentiate MSC into mesodermal cells is preferable, particularly acell with multipotency that can differentiate into mesenchymal cellssuch as osteoblasts, skeletal myoblasts, chondrocytes, adipocytes, bonemarrow stromal cells and smooth muscle cells, by a culture under acondition inducing differentiation into mysenchymal tissues;multipotency that can differentiate into myocardial cells by a cultureunder a condition inducing differentiation into cardiac muscle such as acoculture with cultured myocardial cells; multipotency that candifferentiate into endothelial cells, by a culture under a conditioninducing differentiation into endothelium, such as a culture under acondition maintaining endothelial cells; as well as multipotency thatcan differentiate into neurons that are ectodermal cells, by a cultureunder a condition inducing differentiation into nerve, such as acoculture with cultured neuron.

Furthermore, the present invention relates to a mesodermal progenitor, amesodermal cell or a mesodermal tissue induced to differentiate from theMOMCs of the present invention under induction condition known todifferentiate MSC to mesodermal cells; for instance a mesenchymalprogenitor, a mesenchymal cell or a mesenchymal tissue such asosteoblasts, skeletal myoblasts, chondrocytes and adipocytes, induced bya culture under a condition inducing differentiation of MOMCs intomesenchymal tissues; a myocardial progenitor, a myocardial cell, or amyocardial tissue induced by a culture under a condition inducingdifferentiation of MOMCs into cardiac muscle such as a coculture withcultured myocardial cells; an endothelial progenitor, an endothelialcell or an endothelial tissue induced by a culture under a conditioninducing differentiation of MOMCs into endothelium, such as a cultureunder a condition maintaining endothelial cells; as well as anectodermal neural progenitor, a neuron or a nerve tissue induced by aculture under a condition inducing differentiation of MOMCs into nerve,such as a coculture with cultured neuron.

The osteoblasts induced by culturing under a condition inducingdifferentiation into osteoblasts, are preferably cells with acylinder-like form, being Alizarin red positive for calcium deposition,alkaline phosphatase staining positive, showing increase ofintracellular Ca concentration, and expressing bone sialoprotein II,osteocalcin gene specific to osteoblasts. The chondrocytes induced by aculture under a condition inducing differentiation into chondrocytes arepreferably cells slightly large with a cylinder shape, rich ofcytoplasm, and expressing type II and X collagens, which are specific tochondrocytes. The skeletal muscle cells induced by culturing under acondition inducing differentiation into skeletal muscle cells, arepreferably cells with a long spindle shape, expressing skeletalmuscle-specific actin and myocin. The adipocytes induced by a cultureunder a condition inducing differentiation into adipocytes arepreferably cells with lipid droplets for Oil-red staining, wherein PPARγand aP2 genes expression is enhanced.

As for the myocardial progenitors or myocardial cells induced by aculture under a condition inducing differentiation into myocardial,cells expressing Nestin, myocardial-specific transcription factorsNkx2.5 and eHAND, and myosin light chain gene, a myocardialcell-structural protein, can be preferably exemplified. As for theneural progenitors or neurons induced by a culture under a conditioninducing differentiation into nerve, cells expressing Nestin, andneuron-specific transcription factors NeuroD, Neurogenin 2, and thenexpressing Hu, and NeuN being nerve markers can be preferablyexemplified. As for the endothelial progenitors or endothelial cellsinduced by a culture under a condition inducing differentiation intoendothelium, cells with a polymorphic form with small projections,expressing endothelial-specific marker protein, KDR, vWF and eNOS can bepreferably exemplified.

As described in the above, MOMCs have a differentiation potential intomesodermal cells such as mesenchymal cells under induction conditionknown to mainly differentiate MSC into mesodermal cells, as well as adifferentiation potential into neurons of ectodermal system induced by aculture under a condition inducing differentiation of MOMCs into nerve.Moreover, the differentiation of MOMCs into mesodermal cells such asindividual mesenchymal cells follows the steps observed in MSCdifferentiation, in terms of the timing of lineage-specifictranscription factor expression. For example, the expression ofCialoprotein II and osteocalcin follows the expression of Cbfa1/Runx2(Cell, 108, 17-29, 2002), and further, the expression of MyoD precedesthe expression of SkM-actin and myosin (Front Biosci., 5, D750-767,2000). These findings suggest that differentiation processes intoindividual mesenchymal lineages are shared by MOMCs and MSCs.

As for the preparation method of MOMCs of the present invention, thereis no specific limitation as long as it is a method comprising culturingperipheral blood mononuclear cells (PBMCs) in vitro on fibronectin, forexample on plastic plates coated with fibronectin preferably for 5 to 14days, more preferably for 7 to 10 days, and collecting fibroblast-likecells expressing CD14 and CD34, but it is preferable to culture PBMCswithout any additional growth factors. Collagen or laminin can be usedinstead of the above-mentioned fibronectin, but the differentiation ofmonocytes into MOMCs requires soluble factors derived from CD14⁻ cellsbeside fibronectin. The origin of the above PBMCs is not particularlylimited including experimental animals such as mice, rats, dos, pigs andmonkeys or human, while human PBMCs can be preferably exemplified.Moreover, human PBMCs can be isolated from human venous blood by commonmethods. The above-mentioned culturing method is not limited, and aculturing method comprising culturing at 37° C., with 5% CO₂ in ahumidified atmosphere, at a density of 10⁴ to 10⁷/ml, for example at2×10⁶/ml, and depleting non-adherent cells and supplementing a freshmedium every 2 to 4 days, more preferably every 3 days. Thus obtainedMOMCs of the present invention can be expanded in culture withoutloosing their original phenotype for up to 5 passages.

The present invention relates to a therapeutic agent comprising asactive ingredient the MOMCs of the present invention and/or mesodermalprogenitors, mesodermal cells and/or mesodermal tissues induced todifferentiate from the MOMCs, for instance, mesenchymal progenitors suchas osteoprogenitors, skeletal muscle progenitors, chondroprogenitors,adipoprogenitors, mesenchymal cells such as osteoblasts, skeletalmyoblasts, chondrocytes and adipocytes, mesenchymal tissue such as bone,cartilage, muscle and fat, induced by culturing MOMCs under a conditioninducing differentiation into mesenchymal tissues; myocardialprogenitors, myocardial cells or myocardial tissues induced by culturingMOMCs under a condition inducing differentiation into cardiac muscle,such as a coculture with cultured myocardial cells; endothelialprogenitors, endothelial cells, endothelial tissues induced by culturingMOMCs under a condition inducing differentiation into endothelium suchas a culture under a condition maintaining endothelial cells. Thepresent invention also relates to a therapeutic agent comprising asactive ingredient the MOMCs of the present invention and/or neuralprogenitors, neurons and/or nerve tissues induced to differentiate fromthe MOMCs. Further, it relates to a treating method comprisingadministering the MOMCs of the present invention and/or the mesodermalprogenitors, mesodermal cells and/or mesodermal tissues, the neuralprogenitors, neurons and/or nerve tissues induced to differentiate fromthe MOMCs, for example administering directly to an impaired or deletedsite or to its proximity or to the peripheral blood. It is preferable todetermine appropriately either of MOMCs or MOMCs treated by inducingdifferentiation are suitable for the therapeutic agent, according to thetype of cells or diseases, or administering method. Further, as MOMCsare cells relatively easy to transfer genes, it can be used for tissuereconstitution therapy after introducing a particular gene before celltransplantation to human. For example, when there is some osteogenicimpairment in a certain congenital disease, it is possible to transplantafter modifying the gene or to prepare it to generate a particularprotein (cytokine, growth factor, hormone, etc.).

As described above, as MOMCs have a potential to differentiate intomesodermal tissue such as various mesenchymal tissues or nerve tissues,they are useful as a source of cells for tissue regenerating therapy tocongenital diseases, degenerative diseases, and injuries. For instance,as for disease or pathology to be the object of the therapeutic agent ortreating method of the present invention, bone destruction due todegenerative disease such as dysostosis, fracture, rheumatoid arthritis;cartilage destruction due to rheumatoid arthritis or osteoarthritis, ormuscular disease due to congenital disease such as dystrophy or acquireddisease such as myositis; myocardial disease due to myocardialinfarction or cardiomyopathy, brain disorder such as brain infarction orParkinson disease; injury such as spinal cord damage, or vasculardisease due to arteriosclerosis or connective tissue disease. Moreover,plastic surgery such as breast augmentation and the like is encompassedin the therapeutic agent or treating method of the present invention forconvenience. In cell therapy using MOMCs or MOMCs treated by inducingdifferentiation, there are considerable advantages over currentlyproposed regenerative treatment using tissue-specific stem cells and EScells. In other words, as a large number of monocytes can be obtainedfrom patients by collecting their blood, a minimally invasive procedure,circulating monocytes could be a relatively easy source of autologouscells. Furthermore, the generation of MOMCs from monocytes istechnically easy and quick, and the ethical dilemma of using ES cellscan be bypassed.

The present invention will be explained in detail in the following, butthe technical scope of the present invention will not be limited tothese examples.

METHODS AND MATERIALS Example 1 MOMC Cultures

PMBCs were isolated from heparinized venous blood obtained from healthyadult by Lymphoprep (Nycomed Pharma AS) density gradient centrifugation.All blood samples were obtained after the subjects gave their writteninformed consent, approved by the Institutional Review Boards. Isolatedcells were washed twice with phosphate-buffered saline (PBS) andsuspended in low-glucose DMEM supplemented with 10% heat-inactivated FBS(JRH Biosciences). PMBCs were cultured at a density of 2×10⁶/ml onplastic plates coated with fibronectin without any additional growthfactors at 37° C. with 5% CO₂, in a humidified atmosphere. Three daysafter the culture, non-adherent cells were removed. The medium waschanged to a fresh one every three days and the cells were cultured forup to 4 weeks. After 7-10 days of culture, the adherent cells werecollected as MOMCs and used in the following assays or were moved on newfibronectin-coated plates and maintained in the same culture conditionfor up to 10 passages.

To examine the origin of MOMCs, PBMCs depleted of CD14⁺, CD34⁺ orCD105/endoglin/SH2⁺ cells were cultured on fibronectin-coated plates for7 days. The depletion of CD14⁺ or CD34⁺ cells was performed by using ananti-CD14 or anti-CD34 monoclonal antibody coupled to magnetic beads(DynaBeads) followed by magnetic separation according to themanufacturer's protocol. CD105⁺ cell-depleted PBMCs were prepared byincubating PBMCs with anti-CD105 mAb (Immunotech) and subsequently withgoat anti-mouse IgG antibody coupled to magnetic beads (Dynal).Mock-treated PBMCs incubated with isotype-matched mouse mAb andbead-conjugated anti-mouse IgG antibody were also prepared as a control.The proportion of CD14⁺ cells in the CD14⁺ cell-depleted PBMC fractionwas consistently <0.5%, and the proportions of CD34⁺ cells in the CD34⁺cell-depleted fraction and of CD105⁺ cells in the CD105⁺ cell-depletedfraction were <0.01% by flow cytometry. The number of attaching cellsper cm³ was counted and the results were expressed as the ratio to theuntreated PBMC culture.

Some experiments were performed to separate circulating CD14⁺ monocytesand CD14⁻ cells from PBMCs by using anti-CD14 mAb-coupled magnetic beads(CD14 MicroBeads; Miltenyi Biotech) followed by MACS column separationaccording to the manufacturer's protocol. Flow cytometric analysisrevealed that monocyte and CD14⁻ cell fractions contained >98% and <0.5%CD14⁺, respectively. Monocytes were labeled with PKH67 green (Sigma) andcocultured with unlabeled CD14⁻ cells (ratio of 1:4) onfibronectin-coated plastic plates for 7 days. PKH67-labeled monocyteswere also cultured alone on fibronectin-coated or uncoated plasticplates for 7 days.

Example 2 Preparation of Macrophages and Dendritic Cells

Macrophages were prepared by culturing adherent PMBCs on plastic platesin M199 medium supplemented with 20% FBS and 4 ng/ml M-CSF (R&D Systems)for 7 days (Differentiation, 65, 287-300, 2000). Mature monocyte-deriveddendritic cells were obtained from plastic adherent PBMCs by maturationusing a series of culture conditions (J Immunol Methods, 196, 121-135,1996). Briefly, adherent cells were cultured in RPMI1640 supplementedwith 10% FBS containing 50 ng/ml GM-CSF and 50 ng/ml IL-4 (all fromPeproTech) for 7 days. Next, the immature dendritic cells were incubatedwith 50 ng/ml TNF-α (PeproTech) for 3 days. Flow cytometric analysisrevealed that the macrophage fraction contained 98% or more ofCD14⁺CD80⁺ cells and the dendritic cell fraction contained 95% or moreof CD83⁺HLA-DR⁺ cells and less than 1% of CD14⁺ cells.

Example 3 Cell Lines

Primary cultures of human dermal fibroblasts were established fromdermal biopsies of healthy donors and maintained in low-glucose DMEMsupplemented with 10% FBS. Primary human myoblasts were prepared byculturing muscle biopsies from patients that are histologically normaleven being clinically suspected as myositis (J Cell Biol., 144, 631-643,1999). The human osteosarcoma cell line MG-63, the rhabdomyosarcoma cellline RD, and the chondrosarcoma cell line OUMS-27 were obtained fromHealth Science Research Resources Bank of Japan (Osaka, Japan) andmaintained in low-glucose DMEM supplemented with 10% FBS.

Example 4 In Vitro Differentiation of MOMCs into Mesenchymal Cells

MOMCs which were either freshly generated from PBMCs, cultured forseveral passages or cryo-preserved, were moved on new fibronectin-coatedplastic plates or chamber slides and grown to semi-confluence inhigh-glucose DMEM supplemented with 10% FBS (Hyclone Laboratories).Next, the cells were then cultured under conditions known to induce MSCsto differentiate into various mesenchymal cell types cited in thefollowing (Science, 284, 143-147, 1999; J Cell Biol., 144, 631-643,1999; J Cell Biochem., 64, 295-312, 1997; Muscle Nerve, 18, 1417-1426,1995; Tissue Eng., 4, 415-428, 1998; Arthritis Rheum., 44, 85-95, 2001;J Biol Chem., 275, 9645-9652, 2000). Monocytes, macrophages, and dermalfibroblasts newly isolated from PBMCs were treated under identicalconditions as controls.

As for osteogenesis, the adherent cells were cultured in Osteogenesisinduction medium (Clonetics) containing 100 nM dexamethasone, 10 mMβ-glycerophosphate, and 50 μM ascorbic acid. The medium was changedtwice a week for 3 weeks.

As for myogenesis, the adherent cells were treated with 10 μM5-azacytidine (Sigma) for 24 hours. The cells were washed with PBS andcultured in a medium containing 5% horse serum (Life Technologies), 50mM hydrocortisone (Sigma) and 4 ng/ml basic fibroblast growth factor(Sigma). The medium was changed twice a week for 3 weeks.

As for chondrogenesis, the adherent monolayer cells were cultured for 3weeks in serum-free medium in the presence of TGF-β (R & D systems),which was added to the culture medium every other day so that the finalconcentration become 10 ng/ml.

As for adipogenesis, the cells were incubated with adipogenic inductionmedium supplemented with 1 μM dexamethasone, 0.5 mMmethyl-isobutylxanthine, 10 μg/ml insulin, and 100 mM indomethacin (allfrom Sigma). After 72 h, the medium was changed to maintenance mediumsupplemented with only 10 μg/ml insulin and rested for 24 hours. Thecells were treated three times with adipogenic induction medium andmaintained in the maintenance medium for an additional week.

Example 5 In Vitro Differentiation of MOMCs into Myocardial Cells

MOMCs were cocultured with cultured myocardial cells derived from Wistarrat fetus for 8 days. The cultured cells were fixed with 4%paraformaldehyde, and then immunostained (DAB staining) withhuman-specific anti-nestin antibody (Chemicon).

After labeling cell membrane with fluoroscent PKH67 (Sigma), MOMCs werecocultured with Wistar rat cultured myocardial cells for 7 to 10 days.The cultured cells were fixed with 4% paraformaldehyde, and doublefluorescently immunostained with anti-Nkx2.5 antibody, anti-eHANDantibody (Santa Cruz), and anti-CD45 antibody (DAKO).

MOMCs were cocultured with Wistar rat cultured myocardial cells for 3,6, 9, and 12 days, and mRNA was extracted. Human specific PCR primers(TGACAAGAACGATCTGAGAG (SEQ ID NO: 1), CAGGTTCTTGTAGTCCAAGT (SEQ ID NO:2)) to myocin light chain (MLC2v) being a myocardial cell structuralprotein were constructed and RT-PCR was performed.

Example 6 In Vitro Differentiation of MOMCs into Neurons

MOMCs were cocultured with Wistar rat cultured neurons for 8 days. Thecultured cells were fixed with 4% paraformaldehyde, and thenimmunostained with human-specific anti-nestin antibody (DAB staining).

After labeling cell membrane with fluoroscent PKH67, MOMCs werecocultured with Wistar rat cultured neurons for 4 days. The culturedcells were fixed with 4% paraformaldehyde, and double fluorescentlyimmunostained with anti-NeuroD antibody (Santa Cruz) and anti-CD45antibody (DAKO).

MOMCs were cocultured with Wistar rat cultured neurons for 3 days. Thecultured cells were fixed with 4% paraformaldehyde, and doublefluorescently immunostained with human-specific anti-nestin antibody andanti-Neurogenin 2 antibody (Chemicon).

After labeling cell membrane with fluoroscent PKH67, MOMCs werecocultured with Wistar rat cultured neurons for 9 to 10 days. Thecultured cells were fixed with 4% paraformaldehyde, and doublefluorescently immunostained with anti-Hu antibody and anti-NeuN antibody(Chemicon).

Example 7 In Vitro Differentiation of MOMCs into Endothelial Cells

MOMCs were cultured in endothelial cell maintenance medium EBM-2(Clonetics) for 7 to 10 days as adherent cells. The cultured cells werefixed with 10% neutral buffered formalin, and immunostained (DABstaining) with anti-CD34 antibody (Calbiochem-Novabiochem), anti-vWFantibody (Dako), anti-eNOS antibody (Becton-Dickinson) andanti-VEGFR2/KDR/Flk-1 antibody (Sigma).

RNA was extracted from MOMCs cultured in EBM-2 medium for 7 days, andreverse-transcribed to cDNA. PCR was performed by using specific primersto Flt-1, VEGFR/2/KDR/Flk-1, CD31, CD144, vWF and CD34, CD45, CD14,GAPDH, expressed in endothelial cells. MOMCs before inducingdifferentiation and RNA derived from cultured umbilical vein endothelialcell (HUVEC) were used as control.

Example 8 Flow Cytometry Analysis

Fluorescent cell staining was performed with the following steps. Theadherent cells were detached from the plastic plates by incubation with2 mM EDTA on ice, and blocked with normal mouse or rat serum for 10 minat under 4° C. The cells were stained with the following mouse mAbs orrat anti-Sca-1 mAb (Cedarlane Laboratories), which were eitherunconjugated or conjugated to FITC, phycoerythrin (PE) or PC5:anti-HLA-DR antibody, anti CD11c antibody (BD Pharmingen),anti-CD11b/Mac-1 antibody, anti-CD14 antibody, anti-CD29 antibody,anti-CD34 antibody, anti-CD44 antibody, anti-CD83 antibody,anti-CD105/endoglin/SH2 antibody, anti-CD117/c-kit antibody(Immunotech), anti-CD34 antibody, anti-CD133 antibody (MiltenyiBiotech), anti-HLA class I antibody, anti-HLA-DR antibody,anti-CD31/PECAM-1 antibody, anti-Flt-1/VEGFR1 antibody,anti-Flk-1/VEGFR2 antibody (Sigma), anti-CD40 antibody, anti-CD54antibody, anti-CD80 antibody, anti-CD86 antibody (Ancell),anti-CD144/VE-cadherin antibody, or anti-type I collagen antibody(Chemicon International). When unconjugated mAbs were used, goatanti-mouse antibody or rat IgG F (ab′)₂ antibody conjugated to FITC orPE (Immunotech) was used as a secondary antibody. For intracellularstaining, the cells were permeabilized and fixed by using IntraPrep™permeabilization reagent (Immunotech). Cells were analyzed on aFACSCalibur flow cytometer (Becton Dickinson) by using the Cell Questsoftware. Visualized cells were identified by gating on forward and sidescatters, and the data are shown as logarithmic histograms or dot-plots.

Example 9 Immunohistochemistry

Slides were coated with monocytes, macrophages, or dendritic cells byusing a cytospin technique, and the remaining cell types were culturedon fibronectin-coated chamber slides, except for samples to be used forfibronectin staining, for which type I collagen-coated slides were usedinstead. The cells were fixed with 10% formalin, and the endogeneousperoxidase activity was suppressed with 3% peroxide for 5 min. Slideswere incubated for 30 min with one of the following mouse mAbs, or ratanti-Sca-1 mAb: anti-CD45 antibody, anti-vimentin antibody,anti-skeletal muscle-specific actin antibody (SkM-actin) (Dako),anti-CD34 antibody (Calbiochem-Novabiochem), anti-type I collagenantibody (Chemicon), anti-type III collagen antibody, anti-fibronectinantibody (Sigma), anti-type II collagen antibody (ICN Biomedicals) oranti-skeletal muscle-specific myosin heavy chain antibody (SkM-MHC)(Zymed Laboratories). The slides were then further incubated withbiotin-labeled anti-mouse antibody and anti-rat IgG antibody. Theantibody-biotin conjugates were detected with a streptavidin-horseradishperoxidase complex (Nichirei) applied for 10 min at room temperature byusing 3,3′-diaminobenzidine as the substrate. Nuclei were counterstainedwith hematoxylin. The negative controls were cells incubated with normalmouse- or rat-IgG antibody (DAKO) instead of the above-mentioned primaryantibody.

Fluorescence double-staining was performed as follows. The cells werefixed with 4% paraformaldehyde, and incubated with goat anti-PEBP2αAantibody or anti-Sox9 polyclonal antibody (Santa Cruz), followed byincubation with AlexaFluor R568 goat-specific IgG antibody (MolecularProbes) and then with FITC-conjugated mouse anti-CD45 mAb (Dako).Similarly, the cells were stained with mouse anti-MyoD antibody (Dako)or anti-peroxisome proliferation-activated receptor γ (PPARγ gene) mAb(Santa Cruz), followed by incubation with tetramethylrhodamineisothiocyanate isomer R-labeled mouse-specific IgG antibody (Dako) andsubsequently with FITC-conjugated anti-CD45 mAb. The cells were examinedwith a confocal laser fluorescence microscope (LSM5 PASCAL; Carl-Zeiss).To enumerate the proportion of cells staining positive for a givenmarker, at least 300 cells per culture were evaluated with a lowmagnification.

Example 10 Uptake of Acetylated LDL (Ac-LDL)

The adherent cells were cultured with 2.5 μg/ml Dil-Ac-LDL (MolecularProbes) for 1 hour, and Ac-LDL uptake was evaluated by flow cytometry.

Example 11 Alkaline Phosphatase Staining

The cells were fixed with 10% formalin and subsequently incubated in asolution containing 0.2 mg/ml naphthol AS-TR phosphate and 0.5 mg/mlFast Red RC (all from Sigma) for 10 min.

Example 12 Intracellular Calcium Detection

To evaluate intracellular calcium deposits, the cells were fixed with10% formalin and stained with 2% alizarin red S (Sigma) for 3 min,followed by extensive wash with distilled water. The intracellularcalcium concentration was measured by using a commercially available kit(Sigma) (J Biol Chem., 275, 9645-9652, 2000). The protein content incell extract was also measured by using the Bradford protein assay kit(Bio-Rad Laboratories) by using bovine serum albumin as a standard. Thecalcium concentration was expressed as microgram per microgram ofprotein content.

Example 13 Oil-Red-O Staining

The cells were fixed with 0.2% glutaraldehyde for 5 min, washed with 60%isopropanol, and covered with 0.1% Oil-red-O (Sigma) for 10 min. Afterwashing with 60% isopropanol and subsequently with distilled water, thecells were counterstained with hematoxylin.

Example 14 Transmission Electron Microscopy

Cultured MOMCs were immediately fixed with 2.5% glutaraldehyde,post-fixed with 2% osmium tetroxide, dehydrated in a series of ethanoland propylene oxide, and embedded in Epoxy resin. The cells werethin-sectioned on a LKB ultratome with a diamond knife. Sections in therange of gray to silver were collected on 150-mesh grids, double-stainedwith uranyl acetate and lead citrate, and examined under a JEOL-1200EXII electron microscope (Jeol).

Example 15 Cell Proliferation Studies

Proliferating MOMCs were detected by BrdU-labeling as describedpreviously (Blood, 71, 1201-1210, 1988). It is explained briefly in thefollowing. MOMCs were cultured in the presence of 10 μM BrdU (Sigma) for2 hours before staining. After a 30-minute fixation in Carnoy's fixative(methanol/acetic acid) at −20° C., the cells were air-dried, treatedwith 2N-HCl for 1 hour to denature DNA, and then neutralized with 0.1 Mborate (pH 8.5) for 10 minutes. The cells were then incubated with mouseanti-BrdU mAb (Chemicon International), followed bybiotin-streptavidin-peroxidase complex staining. Nuclei werecounterstained with hematoxylin. Negative controls were the cellsincubated with isotype-matched mouse control mAb instead of theabove-mentioned primary antibody. Apoptotic cells were detected byincubating unfixed cells with propidium iodide (Sigma) for 30 min, andobserved under a fluorescent microscope.

For cell-division studies, the cells were labeled with5-carboxyfluorescein diacetate succinimidyl ester (CFSE) as describedpreviously (J Exp Med., 183, 2313-2328, 1996). CFSE-labeled monocyteswere cocultured with unlabeled CD14⁻ cells on fibronectin-coated platedfor 1, 3 and 5 days, and the adherent cells were harvested and stainedwith PC5-labeled anti-CD14 mAb. CFSE-labeled MOMCs were also culturedfor 1, 3 and 5 days. The color intensity of CFSE labeling was evaluatedby flow cytometry.

Example 16 RT-PCR

Total RNA was extracted from MOMCs that had or had not been induced todifferentiate by using the RNeasy kit (Qiagen). Total RNA was alsoextracted from peripheral blood CD14⁺ monocytes and macrophages,dendritic cells, dermal fibroblasts, myoblasts, and various culturedcells including osteosarcoma, rhabdomyosarcoma, and chondrosarcoma.Human muscle- and fat tissue-derived total RNAs were purchased fromClonetech Laboratories. Single-strand cDNA was synthesized from thetotal RNA by using Molony murine leukemia virus reverse transcriptase(Takara) with oligo-dT as a primer. The cDNA (equivalent to 50 ng totalRNA) was then subjected to PCR amplification by using various specificprimers listed in Table 1, shown by SEQ ID NOs: 3 to 34. The PCRproducts were resolved by electrophoresis on 2% agarose gels andvisualized by staining with ethidium bromide.

TABLE 1 Product Gene Primer sequences size (bp) Osterix Sense:5′-CTTGTGCCTGATACCTGCACT-3′ 470 (SEQ ID NO: 3) Antisense:5′-TCACTCTACCTGACCCGTCATC- 3′ (SEQ ID NO: 4) Bone Sense:5′-AAACGGCACCAGTACCAACA 3′ 394 sialoprotein (SEQ ID NO: 5) II Antisense:5′-GCCATCGTAGCCTTGTCCTT- 3′ (SEQ ID NO: 6) Osteocalcin Sense:5′-GGCAGCGAGGTAGTGAAGAGAC-3′ 257 (SEQ ID NO: 7) Antisense:5′-GGCAAGGGGAAGAGGAAAGAAG-3′ (SEQ ID NO: 8) SkM-MHC Sense:5′-ATAGGAACACCCAAGCCATC-3′ 599 (SEQ ID NO: 9) Antisense:5′-TTTGCGTAGACCCTTGACAG- 3′ (SEQ ID NO: 10) Myogenin Sense:5′-TGGCCTTCCCAGATGAAACC-3′ 452 (SEQ ID NO: 11) Antisense:5′-GCATCGGGAAGAGACCAGAA-3′ (SEQ ID NO: 12) α1 (II) Sense:5′-TTCAGCTATGGAGATGACAATC-3′ 472 collagen (SEQ ID NO: 13) Antisense:5′-AGAGTCCTAGAGTGACTGAG- 3′ (SEQ ID NO: 14) α1 (X) Sense:5′-AATCCCTGGACCGGCTGGAATTC-3′ 267 collagen (SEQ ID NO: 15) Antisense:5′-TTGATGCCTGGCTGTCCTGGACC-3′ (SEQ ID NO: 16) PPARγ Sense:5′-AGGAGCAGAGCAAAGAGGTG- 3′ 474 (SEQ ID NO: 17)Antisense:5′-AGGACTCAGGGTGGTTCAGC-3′ (SEQ ID NO: 18) aP2 Sense:5′-TATGAAAGAAGTAGGAGTGGGC-3′ 290 (SEQ ID NO: 19) Antisense:5′-CCACCACCAGTTTATCATCCTC-3′ (SEQ ID NO: 20) CD34 Sense:5′-CCTCCCAAGTTTTAGGACAA-3′ 362 (SEQ ID NO: 21) Antisense:5′-CAGCTGGTGATAAGGGTTAG-3′ (SEQ ID NO: 22) CD45 Sense:5′-AACCTGAAGTGATGATTGCTG- 3′ 500 (SEQ ID NO: 23) Antisense:5′-TACCTCTTCTGTTTCCGCAC-3′ (SEQ ID NO: 24) CD14 Sense:5′-CTGCGTGTGCTAGCGTACTC-3′ 655 (SEQ ID NO: 25) Antisense:5′-CGTCCAGTGTCAGGTTATCC-3′ (SEQ ID NO: 26) Cbfa1/Runx2 Sense:5′-GTCTTACCCCTCCTACCTGA-3′ 183 (SEQ ID NO: 27) Antisense:5′-TGCCTGGCTCTTCTTACTGA- 5′ (SEQ ID NO: 28) MyoD Sense:5′-CCTAGACTACCTGTCCAGCATC- 3′ 365 (SEQ ID NO: 29) Antisense:5′-GGCGGAAACTTCAGTTCTCC-3′ (SEQ ID NO: 30) Sox-9 Sense:5′-CCCGATCTGAAGAAGGAGAGC-3′ 380 (SEQ ID NO: 31) Antisense:5′-GTTCTTCACCGACTTCCTCCG- 3′ (SEQ ID NO: 32) GAPDH Sense:5′-TGAACGGGAAGCTCACTGG-3′ 307 (SEQ ID NO: 33) Antisense:5′-TCCACCACCCTGTTGCTGTA-3′ (SEQ ID NO: 34)

Example 17 Statistical Analysis

All comparisons were tested for statistical significance by usingMann-Whitney U test.

RESULTS Example 18 Generation of MOMCs

When PBMCs were cultured on fibronectin-coated plates in DMEMsupplemented only with 10% FBS, a subset of cells immediately attachedto the plates. Small clusters of round cells developed within 24 hours,and cell processes extended from them. After 5 days of culture, adherentcells with a fibroblast-like morphology made their appearance. Over theensuing 3 days, the fibroblast-like cells became the predominant celltype in the culture (FIG. 1 a). The fibroblast-like cells werefrequently detected around the clusters (FIG. 1 b). The number offibroblast-like cells increased slowly until around Day 14. After thistime, the proliferating ability gradually stopped but the cells survivedfor up to 4 weeks. When 10⁸ PBMCs obtained from 50 donors were cultured,0.3 to 1.0×10⁷ adherent cells were obtained at Day 7. From flowcytometric analysis, the cells harvested on Day 7 showed to comprise asingle phenotype (95% or more were homogeneous) and to be positive forCD14, CD45, CD34, and type I collagen (FIG. 2). This phenotype is uniqueand distinct from that of known adherent cells of peripheral bloodorigin, including monocytes/macrophages (CD14⁺, CD45⁺, CD34⁻ and type Icollagen⁻), endothelial progenitors (CD14⁻, CD45⁻, CD34⁺ and type Icollagen⁻) (Science, 275, 964-967, 1997), and mesenchymal progenitors(CD14⁻, CD45⁻, CD34⁻, type I collagen⁺) (Arthritis Res., 2, 477-488,2000). Cells obtained from at least 50 donors showed the same phenotype.After the cells were moved on new fibronectin-coated plates on Day 7 andcultured under the same conditions, nearly all the cells adopted anelongated fibroblast-like morphology (FIG. 1 c). These cells could beexpanded up to 5 passages, and the cell proliferation was likely to bemost active just after the passage. However, after 5 passages, the cellproliferation speed became gradually slow, and the proliferation abilitydisappeared after 8 passages. The cells did not differentiate naturallyinto mature mesenchymal cells during the culture when no particulartreatment was performed. However, when cells were inoculated at a highconfluency, cells with plural nuclei appeared at a very low frequency.These fibroblast-like cells obtained from PBMCs from a culture in vitro,were named MOMC from the following findings.

By electron microscopic examination, it was revealed that MOMCs had aspindle shaped morphology and contained a number of cytoplasmicorganelles (FIGS. 1 d-g). Multiple primary lysosomes, cell surfaceprojections like pseudopodia, and labyrinth-like endocytic vesicles arespecific observations that are also found in macrophages and otherphagocytes. MOMCs also had prominent bundles of intermediate filaments,well-developed secondary lysosomes, and elongated and branchingmitochondria, which are characteristics frequently seen in cells ofmesenchymal origin. Small lipid droplets were observed in almost allMOMCs. In addition, structures similar to rod-shaped micro-tubulatedbodies, which are specific to endothelial cells, were frequentlydetected. These ultrastructural findings represented mixed features ofphagocytes, and mesenchymal and endothelial cells.

Example 19 Origin of MOMCs in Circulation

The adherent cells obtained in the PBMC culture on fibronectin-coatedplates were serially examined for their surface expression of CD14 andCD34 by flow cytometry (FIG. 3 a). The majority of cells attached to theplates at 1 hour were CD14⁺ and CD34⁻, but CD34 expression graduallyup-regulated on the adherent CD14⁺ cells. Nearly all the adherent cellswere positive for both CD14 and CD34 after 7 days in culture. Asperipheral blood contained CD34⁺ endothelial progenitors (Science, 275,964-967, 1997) and CD105/endoglin/SH2⁺ mesenchymal progenitors (Science,284, 143-147, 1999; Arthritis Res., 2, 477-488, 2000; Biochem BiophysRes Commun., 265, 134-139, 1999), the present inventors examined theeffect of depleting PBMCs of cells positive for CD14, CD34, or CD105 onthe in vitro induction of MOMCs. As shown in FIG. 3 b, the appearance ofMOMCs was almost completely inhibited by the depletion of CD14⁺monocytes, whereas depletion of CD34⁺ or CD105⁺ cells showed no effect.To further confirm that MOMCs originated from circulating monocytes,CD14⁺ monocytes were isolated from PBMCs, labeled with PKH67 andcultured with unlabeled CD14⁻ cells on fibronectin-coated plates. Asshown in FIG. 3 c, PKH67-labeled monocytes expressed CD34 after one weekof the culture. Together, these findings indicate that MOMCs originatesfrom circulating CD14⁺ monocytes. However, non-adherent cells collectedon Day 3 contained a significant proportion of CD14⁺ cells, whichsuggests that a subset of CD14⁺ monocytes can attach to fibronectin anddifferentiate into MOMCs.

When PKH67-labeled monocytes were cultured alone on fibronectin, only afew cells became fibroblast-like and CD34 expression was notup-regulated at Day 7 (FIG. 3 c). In this case, it was sufficient to addculture supernatant of CD14⁻ cells to induce CD34 expression inmonocytes. Furthermore, when coculturing CD14⁻ cells with PKH67-labeledmonocytes on plates that are not coated with fibronectin, the number ofcells attached to the plates on Day 7 was low, and these adherent cellsdid not express CD34 (FIG. 3 c). From these results, it is likely thatthe differentiation from circulating monocytes into MOMCs requiressoluble factor (1 or more) derived from CD14⁻ cells in peripheral bloodand binding to fibronectin.

To evaluate whether CD14⁺ monocytes proliferate during MOMCdifferentiation, CD14⁺ monocytes were labeled with CFSE and coculturedwith unlabeled CD14⁻ cells on fibronectin. Adherent cells were seriallyharvested and examined for color intensity of CFSE labeling and CD14expression by flow cytometry (FIG. 3 d). Nearly all fibronectin-attachedmonocytes proliferated mainly during the first 24 hours of culture, andproliferated slowly later in culture. Adherent cells were almostexclusively CFSE-labeled CD14⁺ monocytes, and the expansion of adherentcells from the CD14⁻ cell fraction was not observed, suggesting thatgeneration of MOMCs did not occur as a result from the growth ofspecific precursors in the mixed CD14⁻ cell population.

Example 20 Phenotype of MOMCs

Expression of various cell surface molecules and intracellular moleculesof MOMCs was examined by flow cytometry and immunohistochemistry (FIGS.2 and 4), and compared the protein expression profile with that ofmonocytes, macrophages, and dendritic cells (Table 2). MOMCs expressedhematopoietic and monocyte lineage marker genes (CD45, CD14, CD13,CD11b, CD11c, and CD64), but did not express dendritic cell marker genes(CD1a and CD83). The expression of HLA Class I, HLA-DR, andcostimulatory molecules (CD40 and CD86) on MOMCs strongly suggests thatMOMCs have an ability to induce antigen-specific T cell activation asantigen-presenting cells. MOMCs expressed hematopoietic stem/endothelialcell marker gene CD34, and mesenchymal stem/endothelial cell marker geneCD105/endoglin/SH2 (Biochem Biophys Res Commun., 265, 134-139, 1999).Moreover, MOMCs expressed stem cell marker gene Sca-1, but did notexpressed different stem cell marker genes CD117/c-kit and CD 133.Further, MOMCs were positive for the endothelial marker geneCD144/VE-cadherin and Flt-1/VEGFR1, while Flk-1/VEGFR2 and vWFexpressions were not observed. MOMCs were also positive for type I andIII collagen, fibronectin, and vimentin, which are extracellular matrixproteins typically produced by cells of mesenchymal origin. Theseprotein expression profiles did not change for up to 5 passages. MOMCsshowed distinct phenotypes from that of monocytes and monocyte-derivedphagocytes. In particular, the expression of stem cell marker genes(CD34, Sca-1 and CD105), endothelial marker genes (CD144/VE-cadherin andFlt-1/VEGFR1), and mesenchymal marker genes (type I and III collagen,and fibronectin) is unique characteristic of MOMCs. Therefore, it can berecognized that MOMCs are cells with phenotypes of phagocytes,endothelial cells, mesenchymal cells and stem cells.

TABLE 2 Mono- Macro- Dendritic cytes phages cells MOMCs CD45 ++ ++ + +Monocyte markers CD14 ++ ++ − ++ CD13 + + + ++ CD11b/Mac-1 ++ + ++ +CD11c + + + + CD64 + + − + Dendritic cell markers CD1a − − −/++^(A) −CD83 − − ++ − HLA molecules HLA class I ++ +++ +++ ++ HLA-DR ++ ++ ++ ++Costimulatory molecules CD40 + + ++ + CD80 − ++ ++ − CD86 + + +++ +Adhesion molecules CD29 + + + + CD44 ++ ++ ++ +/++^(A) CD54 + + ++ +Stem cell/progenitor markers CD34 − − − + CD105/endoglin/SH2 − − − +CD117/c-kit − − − − CD133 − − − − Sca-1 − − − +/++^(A) Endothelial cellmarkers CD31 + + + + CD144/VE-cadherin − − − + Flt-1/VEGFR1 − − − +Flk-1/VEFR2 − − − − vWF^(A) − − − − Ac-LDL + ++ − ++ Mesenchymal cellmarkers Type I collagen − − − + Type III collagen − − − + Fibronectin −− − + Vimentin + + + ++

Example 21 Proliferating Ability of MOMCs

MOMCs seemed to increase during culture. To investigate whether thisobservation is due to cell division, the proportion of dividing cells inMOMCs was evaluated serially by BrdU staining (FIG. 5 a). Nearly half ofthe adherent cells were stained with BrdU after 1 day from the passage,but the number of cells of BrdU⁺ cells decreased significantly at Day 5.The proportion of BrdU⁺ cells to all adherent cells were calculated atDay 1, 3, 5, 7 and 10, and the proportion of BrdU⁺ cells were at themaximum at Day 1 during culture, and decreased chronologicallyafterwards. Cells positive for propidium iodide staining were less than1% at all time points. By investigating using CFSE labeling, it appearedthat MOMCs divided actively and synchronously after the passage and nosubset of the cells proliferated predominantly (FIG. 5 c). Thesefindings suggest that MOMCs have proliferating ability during culture,and that mainly proliferate just after the passage.

Example 22 In Vitro Differentiation of MOMCs into Mesenchymal CellLineages

As MOMCs had some morphologic and phenotypic properties of mesenchymalcells, the present inventors hypothesized that MOMCs can be induced todifferentiate into some mesenchymal lineages. To confirm thishypothesis, MOMCs were cultured under various conditions known to inducedifferentiation of MSCs into bone, skeletal muscle, cartilage and fat.

MOMCs treated with the osteogenic induction procedure underwent a changein their morphology from spindle-shaped to cylinder-like form. It wasobserved that almost every adherent cell formed calcium deposits, byalizarin red staining (FIG. 6 a), and these adherent cells expressedalkaline phosphatase (FIG. 6 b). The intracellular calcium content wassignificantly increased during this process (FIG. 6 c). After osteogenicinduction, MOMCs expressed mRNAs for bone sialoprotein II produced bymature osteocytes and osteocalcin (Calcif Tissue Int. 62, 74-82, 1998)and for bone-specific transcription factor osterix (Cell, 108, 17-29,2002). On the other hand, CD34, CD45, and CD14 expression was lost afterthis induction treatment (FIG. 6 d).

When MOMCs were treated with 5-azacytidine and cultured under acondition inducing differentiation into muscle for 3 weeks, the cellsbecame elongated, but no cells like myocytes with plural nucleiappeared. At this time point, expression of SkM-actin and SkM-MHC wasinduced in 45-60% of the adherent cells, depending on the sample (FIGS.6 e and 6 f). By RT-PCR, mRNAs for the muscle-specific transcriptionfactor myogenin and SkM-MHC were detected after induction (FIG. 6 g).The expression of CD34, CD14, and CD45 was reduced but not lost.Immunohistochemical analysis revealed that CD34 was expressed in nearlyall adherent cells, but CD14 and CD45 were expressed in cells that didnot express SkM-MHC (data not shown). The expression CD34 was alsodetectable in cultured myoblasts and muscle tissue, and even in arhabdomyosarcoma cell line, consistent with the expression of CD34 in asubset of primitive muscle cells (J Cell Biol., 150, 1085-1100, 2000).

To induce cartilage formation, MOMCs were cultured in micromasssuspension in the presence of TGF-β1, which is a standard method toinduce chondrocyte differentiation in MSC (Science, 284, 143-147, 1999;Tissue Eng., 4, 415-428, 1998; Arthritis Rheum., 44, 85-95, 2001).However, MOMCs died within 1 week even in culture according to severaldifferent protocols by droplet-micromass on plates, or pelette-macromassin conical tube. Therefore, monolayer MOMCs were cultured in thepresence of TGF-β1 for 3 weeks. As it is shown in FIG. 6 h, type IIcollagen typical of articular cartilage, was weakly expressed inuntreated MOMCs, but its expression was markedly up-regulated afterinduction treatment. Results of RT-PCR further demonstrated theup-regulated expression of chondrocyte-specific type II and type Xcollagen after the induction treatment (FIG. 6 i). The expression ofCD45 and CD14 was lost, but CD34 expression was retained after theinduction treatment. On the other hand, CD34 was also expressed in achondrosarcoma cell line.

Electron microscopic examination revealed small lipid droplets in MOMCs(FIGS. 1 d, f and g). After the induction treatment, lipid vacuolesappeared and increased over time in both size and number. These lipidvacuoles were stained with Oil-red-O (FIG. 6 j). From this inductiontreatment, 50 to 80% of the adherent cells were committed to thislineage, depending on the sample. mRNAs for PPARγ genes, and mRNAs forthe fatty acid binding protein aP2 were weakly expressed in MOMCs, butthe expression of these genes were markedly up-regulated after theabove-mentioned induction treatment (FIG. 6 k). Expression of mRNAs forCD45 and CD14 was lost, but CD34 expression was retained after theinduction treatment. On the other hand, CD34 was also expressed in fattissues.

The differentiation into mesenchymal cells was observed in MOMCs thatwere freshly generated from PBMCs, cultured for up to five passages orcryo-preserved. In addition, MOMCs obtained from 5 donors showed similardifferentiation potential. Two strains of human dermal fibroblasts,which are mature mesenchymal cells as well as freshly isolated CD14⁺monocytes and macrophages, were also cultured under the identicalinduction conditions. After 3 weeks, the dermal fibroblasts did not showany differentiation tendency in these conditions, although the cellsappeared healthy. After 1 week, circulating monocytes and macrophagessubjected to these culture conditions detached from the plates withoutapparent differentiation. Lineage-specific differentiation was observedin more than half of the adherent cells after 3 weeks of culture. Butthe number of the adherent cells decreased for 20 to 50% from theinitial number of cells, suggesting that most MOMCs were detached duringthe induction process.

To investigate whether the differentiation into a certain lineage isspecific to various induction treatments, cultures from each treatmentwere cross-stained with alizarin red, Oil-red-O, and were alsoimmunostained with SkM-MHC or type II collagen. These cells werepositive only for the staining specific to the intended lineage, andnegative to all other stainings (data not shown). Furthermore, toinvestigate the possibility that some subsets differentiate into lineageother than the intended lineage in an in vitro assay, cells treated withdifferentiation induction for 3 weeks were subjected to high-sensitiveRT-PCR, to amplify the transcripts of plural genes wherein theexpression is limited to osteogenic (osteocalcin), myogenic (SkM-MHC),chondrogenic (type II collagen), or adipogenic (aP2) lineage. Expressionof lineage-specific gene was specific to the intended lineage (data notshown), suggesting that the differentiation was exhibited specificallyto the performed treatment.

To exclude the possibility that the differentiated mesenchymal cellswere derived from cells with differentiation potential as MSCcontaminated into MOMC fractions, CD14⁺ cells were positively purifiedfrom the MOMCs by using the MACS separation system before the inductiontreatment. As expected, the differentiation into the osteogenesis,myogenesis, chondrogenesis, and adipogenesis was observed as for theunselected MOMCs. In addition, the depletion of cells expressing CD34 orCD105/endoglin/SH2 at the initiation of PBMC cultures did not affect thedifferentiation into mesenchymal cells. Further the expression oflineage-specific transcription factors, Cbfa1/Runx2 (Cell, 89, 755-764,1997), MyoD (Front Biosci., 5, D750-767, 2000), Sox-9 (OsteoarthritisCartilage, 8, 309-334, 2000), and PPARγ (J Biol Chem., 276, 37731-37734,2001), in MOMCs after 1 week of induction treatment, was examined. As atthis time point, as MOMCs were expressing CD45, MOMCs that underwentosteogenic, myogenic, chondrogenic, or adipogenic induction treatmentwere double-stained, to investigate the expression of CD45 andindividual transcription factors. As shown in FIG. 7, MOMCs thatunderwent osteogenic differentiation for 1 week, expressed CD45 in cellmembrane and cytoplasma, and Cbfa1/Runx2 in nucleus. Similarly,simultaneous expression of CD45/MyoD and CD45/Sox-9 was observed inMOMCs that underwent induction of myogenic and chondrogenicdifferentiation for 1 week. PPARγ was weakly expressed in the nuclei ofuntreated MOMCs, but after 1 week of adipogenic induction, the PPARγexpression increased and the CD45 expression decreased. These findingssuggest that CD45⁺ hemapoietic cells underwent lineage-specificdifferentiation under specific conditions inducing differentiation. Theexpression of these lineage-specific transcription factors, exceptPPARγ, was lost at 3 weeks of the induction treatment as determined byimmunohistochemistry and RT-PCR (data not shown). These findings suggestthat MOMCs express transiently the above-mentioned lineage-specifictranscription factors in the primary differentiation step of theinduction treatment.

Example 23 In Vitro Differentiation of MOMCs into Myocardial Cells

Nestin (brown), a marker expressing in nerve and myocardial progenitors,was expressed in MOMCs at Day 8 of coculture, and binding withsurrounding rat-cultured myocardial cells was observed (FIG. 8A).

MOMCs labeled with PKH67 (Green) expressed myocardial cell-specifictranscription factors Nkx2.5, eHAND (Red/Alexa568: Molecular Probe), andexpressed simultaneously CD45, a hematopoietic marker (Blue/Alexa660:Molecular Probe) (FIGS. 8B, C). This shows the differentiating processof MOMCs derived from human peripheral blood hemocyte into myocardialcells.

By RT-PCR using human-specific PCR primer, expression of myocin lightchain (MLC2v) which is a myocardial cell structural protein was observedin human cardiac muscle, the positive control, while no expression wasobserved in rat-cardiac muscle, the negative control, nor in MOMCsbefore coculture. In MOMCs at Day 12 of coculture, expression of humanMLC2v was observed (FIG. 8D).

From these results, with the coculture of MOMCs with rat-myocardialcells, induction of differentiation of MOMCs into myocardial cells wasdemonstrated, as well as the expression of myocardial progenitor markerat Day 8-10 of coculture, and the expression of myocardial structuralprotein at Day 12-14.

Example 24 In Vitro Differentiation of MOMCs into Neurons

Nestin (brown), a marker expressing in nerve and myocardial progenitors,was expressed in MOMCs at Day 8 of coculture, and elongation of nerveprojection toward surrounding rat-cultured neurons was observed (FIG.9A).

MOMCs labeled with PKH67 (Green) expressed a neuron-specifictranscription factor NeuroD (Red/Alexa568: Molecular Probe) at Day 4 ofcoculture, and expressed simultaneously CD45 a hematopoietic marker(Blue/Alexa660: Molecular Probe) (FIG. 9B). This shows thedifferentiating process of MOMCs derived from human peripheral bloodhematopoietic cells into neurons.

At Day 3 of coculture, at the same time MOMCs expressed nestin(Green/Alexa488: Molecular Probe), Neurogenin2 (red/Alexa568: MolecularProbe), which is a neuron-specific transcription factor was expressed(FIG. 9C). From these results, MOMCs were shown to be a progenitor ofneurons, whose differentiation into neuron is determined.

With 9 days of coculture with Wistar rat-cultured neuron, expression ofmature nerve markers Hu, NeuN (Red/Alexa568: Molecular Probe) wasobserved (FIGS. 9D, and E) in MOMCs labeled with PKH67 (Green). Theseresults demonstrated that MOMCs differentiate up to mature neurons.

Example 25 In Vitro Differentiation of MOMCs into Endothelial Cells

MOMCs that underwent induction of differentiation in EBM-2 medium for 7days, changed their morphology from spindle shape to a morphology havingmultiple projections, and expressed vWF, eNOS, VEGFR2/KDR/Flk-1 specificto endothelial cells that were not expressed originally.

From a gene expression analysis by RT-PCR, in MOMCs that underwentinduction of differentiation in EMB-2 medium for 7 days, expression ofgenes Flt-1, VEGFR2/KDR/Flk-1, CD31, CD144, vWf characteristic toendothelium was observed. Expression of Flt-1 and CD31 was observed inMOMCs, but other genes were expressed after the induction. Afterinducing differentiation, CD34 expression was enhanced, and expressionof CD45, CD 14 was lost.

From these results, it was demonstrated that by culturing MOMCs inmaintenance medium of endothelial cells, differentiation intoendothelial cells was induced.

INDUSTRIAL APPLICABILITY

According to the present invention, a monocyte-derived multipotent cell,MOMC, having a potential to differentiate into various cells such asmesenchymal cells including bone, cartilage, skeletal muscle and fat,endothelial cells, myocardial cells, neurons, very useful to celltherapy or regenerative medicine can be obtained. Moreover, as monocytescan be easily obtained without much invasion from peripheral blood, andmonocytes represent about 20% of the peripheral blood mononuclear cells,sufficient cells necessary can be supplied in a stable manner. Inducingdifferentiation from monocytes into MOMCs can be performed easily,rapidly at a low cost, and no particular device is needed. Further, asautologous cells can be used for cell transplantation, there are noproblems such as securing donors, or rejection symptoms, and fewerethical problems. The present invention challenges the traditional andbiological view regarding the monocyte/phagocyte systems and willcontribute greatly to the understanding of the differentiation potentialof monocytes and the roles they play for retaining homeostatis of theliving body and induction of pathology.

1. An isolated monocyte-derived multipotent cell (MOMC) expressing CD14,CD34, CD45, type I collagen, and HLA-DR, wherein the cell differentiatesinto osteoblasts, skeletal myoblasts or chondrocytes, and themonocyte-derived multipotent cell (MOMC) is obtained by culturingperipheral blood mononuclear cells (PBMCs) in vitro on fibronectin, andcollecting fibroblast-like cells expressing CD14 and CD34.
 2. Theisolated monocyte-derived multipotent cell (MOMC) according to claim 1,that differentiates into mesenchymal cells by a culture under acondition inducing differentiation into mesenchymal tissues.
 3. Theisolated monocyte-derived multipotent cell (MOMC) according to claim 2,wherein the mesenchymal cells are adipocytes.
 4. The isolatedmonocyte-derived multipotent cell (MOMC) according to claim 1, thatdifferentiates into myocardial cells by a coculture with culturedmyocardial cells.
 5. The isolated monocyte-derived multipotent cell(MOMC) according to claim 1, that differentiates into neurons by acoculture with cultured neurons.
 6. The isolated monocyte-derivedmultipotent cell (MOMC) according to claim 1, that differentiates intoendothelial cells by a culture under a condition maintaining endothelialcells.
 7. The isolated monocyte-derived multipotent cell (MOMC)according to claim 1, that differentiates into mesodermal cells.
 8. Amethod for preparing the monocyte-derived multipotent cell according toclaim 1, comprising culturing peripheral blood mononuclear cells (PBMCs)in vitro on fibronectin, and collecting fibroblast-like cells expressingCD14, CD34, CD45, type I collagen, and HLA-DR.
 9. The method forpreparing the monocyte-derived multipotent cell according to claim 8,comprising culturing in vitro on fibronectin for 5 to 14 days.